The goals of this application are to 1) define the molecular components that mediate the ceramide-induced remodeling of cortical F-actin underlying the apical brush border membrane (BBM) of polarized intestinal epithelia, and 2), test how endogenous sphingomyelinase (SMase) activity at the BBM contributes to the regulation of cytoskeletal dynamics or otherwise affects polarized cellular physiology. Our hypothesis is that acute SMase action at the BBM induces ceramide microdomains that alter the spatial distribution and/or activity of upstream regulators of cortical F-actin structure/function. Such regulation has consequences for pathogenic microbes and microbial products that require F-actin for invasion into host cells. The BBM of intestinal epithelia is highly enriched in sphingomyelin (SM), a sphingolipid that coalesces with cholesterol and other membrane components into microdomains termed lipid rafts, which are linked both structurally and functionally to the actin cytoskeleton. I have discovered that the conversion of SM to ceramide in the BBM of human intestinal epithelia induces a striking reorganization of the cortical actin network. How this occurs is not known. Two complementary approaches are proposed to delineate the mechanism(s) by which ceramide generation in the apical plasma membrane (PM) of polarized epithelia induces structural and functional alterations in the cortical actin cytoskeleton: The first approach will test if SMase treatment impacts F-actin structure and function by altering the localization or turnover of phosphatidylinositol (4,5)-bisphosphate (PIP2, a well-known regulator of actin dynamics). We will examine PIP2 dynamics in vivo using a GFP-PH domain reporter transfected into polarized T84 and Caco2 cell models. The functional requirements of PIP2 for initiating the F-actin phenotype will be investigated by pharmacologically inducing/inhibiting PIP2 accumulation. We will also test if other known regulators of polarized cell cortical actin structure/function are responsible for mediating the observed SMase-induced F-actin phenotype by using small molecule inhibitors and activators. The second approach will explore the role of endogenous SMase in modulating lipid raft and/or cortical actin function at the apical PM of polarized epithelia. I recently found that polarized intestinal T84 cells express an alkaline isoform of SMase (alk-SMase) as an apical ecto-enzyme that uses SM in the outer PM leaflet as substrate under steady-state conditions. Does alk-SMase play a role in modulating BBM lipid raft function including the linkage of these microdomains to cortical F-actin? Initial experiments show that the genetically-tractable Caco2 and MDCK polarized cell models also express alk-SMase, so we will silence alk-SMase expression in these cells by shRNA and assay for alterations or defects in the structure and function both of lipid raft microdomains and the F-actin cytoskeleton. PUBLIC HEALTH RELEVANCE: The proposed experiments in this grant will explore how a certain class of lipid-degrading enzyme (sphingomyelinase) affects host intestinal cell physiology. This is particularly relevant to the intestine as numerous bacterial species inhabiting the human gut also make and secrete this class of enzyme. This work is important because it will increase our understanding of how the regulation of lipids in the membranes of human intestinal cells impacts the machinery that is co-opted by pathogenic microbes and microbial products to invade host cells.